C-Myb is a proto-oncogene with sequence similarity to the viral oncogene v-Myb. V-Myb is capable of causing acute myeloid leukemia (AMI) in chickens and can transform hematopoietic cells in vitro. Although v-Myb has been shown to transform cells, c-Myb lacks v-Myb ability to transform. Both transcription factors are able to bind to promoters of target genes such as the mim-1 gene in vitro, but v-Myb cannot activate the endogenous mim-1. This along with other data suggests that the two genes have very distinct specificities, which are influenced by in vivo factors. One mechanism that may contribute to specificity of in vivo transcription factors is post-translational modifications. The following proposed research aims to determine how specific post-translational modifications influence endogenous gene expression throughout the cell cycle of primary human T-cells. Specific aim 1 involves the characterization of post-translational modifications during specific time points in the cell cycle. Quiescent T-cells will be induced with phytohemagglutinin, immunoprecipitated and immunobloted with anti-modification specific antibodies. As a follow up to aim 1, specific aim 2 will analyze the promoter targets for specifically modified versions of c- Myb. Samples will be taken at specific time points in the cell cycle and subjected to chromatin immunopreciptitation. Those enriched samples will then be subjected to Affymetrix tiling arrays to identify c- Myb target genes. The results from the above immunoprecipitations in specific aim 1 can then be correlated with the promoter targets identified in specific aim 2. Together these two aims serve to identify and characterized c-Myb specificity throughout specific time points in the cell cycle. Specific aim 3 is related to the above aims and will lead to further knowledge of the cell cycle. C-Myb has been shown by the laboratory to bind to CDK6, but the time course of this interaction is currently unknown. Specific aim 3 will use specific time points during the cell cycle to analyze at what stage in the cell cycle CDK6 interacts with c-Myb. Flow cytometry with propidium will be used to determine what stage of the cell cycle the cells are in. These experiments will help to determine the influence of post-translational modifications on c-Myb specificity. Examining c-Myb modification specific gene expression in the cell cycle could lead to break throughs in understanding hematopoiesis and many of the unknown processes in hematopoietic differention. This research stands to contribute to the overall understanding of the phenotypic differences between c-Myb and v-Myb, which can enhance our understanding of human acute myeloid leukemia.